Project summary In Parkinson's Disease (PD), the proteostasis network fails to counter the misfolding of ?-synuclein (?-syn). ?- Syn forms soluble toxic oligomers and self-templating amyloid fibrils that can initiate and propagate disease de novo. ?-Syn fibrils cluster into large cytoplasmic inclusions termed Lewy Bodies, a pathological hallmark of PD. Human molecular chaperones, Hsp110, Hsp70, and Hsp40 can disaggregate ?-syn fibrils and reduce their toxicity. These findings together with our advances with Hsp104 and potentiated variants suggest that ?-syn oligomers and fibrils are not intractable but can be rapidly disassembled into non-toxic forms. More recently, a second human protein disaggregase, HtrA1, has been discovered that disassembles and degrades tau and amyloid-beta fibrils. HtrA1 is a chaperone and homo-oligomeric PDZ serine protease found in the cytoplasm and extracellular space, which selectively degrades misfolded substrates while leaving folded substrates alone. Our unpublished data suggest that HtrA1 rescues ?-syn toxicity in yeast, and, disassembles and degrades ?- syn fibrils in vitro. We hypothesize that small-molecule enhancers of HtrA1 disaggregase activity could stimulate the elimination of deleterious ?-syn accumulations in the degenerating neurons of PD patients. Thus, we propose that the unanticipated protein disaggregase activity of HtrA1 represents a promising, novel PD- relevant target. We hypothesize that enhancing the activity of HtrA1 disaggregase activity with specific small molecules will enable dissolution and degradation of toxic oligomeric and amyloid forms of ?- syn, and confer therapeutic benefits in PD. Thus, we will pursue two specific aims: (1) Isolate small molecules that enhance the ?-syn disaggregase activity of HtrA1; and (2) Assess the ability of small-molecule enhancers of HtrA1 disaggregase activity to rescue ?-syn aggregation and toxicity in mammalian primary neuron models of PD. By the end of this project, there will be a clear ?go/no go? decision for moving a small- molecule enhancer of HtrA1 into rodent models and ultimately PD patients. Small-molecule stimulation of HtrA1 disaggregase activity could eliminate deleterious ?-syn misfolding in degenerating dopaminergic neurons and provide a game-changing solution for PD. Importantly, small-molecule enhancers of HtrA1 disaggregase activity may also have important applications in other neurodegenerative disorders caused by deleterious protein misfolding, including Alzheimer's disease and frontotemporal dementia.